CN107561074A - A kind of biological colorimetric sensor, preparation method and its application for detecting glucose - Google Patents
A kind of biological colorimetric sensor, preparation method and its application for detecting glucose Download PDFInfo
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- CN107561074A CN107561074A CN201710892575.2A CN201710892575A CN107561074A CN 107561074 A CN107561074 A CN 107561074A CN 201710892575 A CN201710892575 A CN 201710892575A CN 107561074 A CN107561074 A CN 107561074A
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Abstract
The invention discloses a kind of biological colorimetric sensor, preparation method and its application for detecting glucose, preparation method is：The mixture of glucose solution and glucose oxidase solution is added in cushioning liquid, cultivates, then adds methylene blue solution, mixes, adds ssDNA cushioning liquid, mixed solution is made；Produce biological colorimetric sensor.Compared with prior art, the preparation method of glucose sensor provided by the invention, cost is low, and design is simple, and the glucose of various concentrations can make solution colour change therefore easily with the naked eye read.Interacted by methylene blue and single stranded DNA and utilize the Conformation Transition for the single stranded DNA for carrying poly- cytimidine, the sensor of detection glucose can be prepared.As a result show that this sensor has sensitive detection for 5 μM to concentration of glucose to 0.8mM, and there is simple to operate, high sensitivity, test limit is low.
Description
Technical field
The invention belongs to biology sensor preparing technical field, and in particular to a kind of biological colorimetric sensor, preparation method
And its application of detection glucose, be it is a kind of interact and utilize based on methylene blue and single stranded DNA carry poly- cytimidine
The glucose sensor of the Conformation Transition structure of single stranded DNA, and glucose can be detected.
Background technology
Fuel of the glucose as generally existing in organism metabolic pathway, is the basic need for meeting vital movement.One
Individual uncommon glucose body fluid levels are a caution signals of human health.For example, on clinical medicine, diabetes are serious
The health of the mankind is endangered, it is diagnosed and treatment is always a great difficult problem of medical field.By to blood sugar in diabetic patients
The accurate measurement of content, effectively diabetes can be monitored and be treated.As can be seen that blood sugar monitoring is to assessing healthy shape
Condition is extremely important.
Since 1962 propose first glucose sensor, the glucose sensor prepared using various technologies is had
Very big development.However, existing glucose sensor is related to complicated and time-consuming tediously long detection process more, there is low choosing more
The shortcomings of selecting property and of a relatively high detection limits, limit their actual applicability.
Therefore, it is a growing need to develop high sensitivity, selectivity glucose sensor high, simple to operate
Ask.It is significant to explore new method detection glucose.
The content of the invention
In order to solve the above technical problems, the invention provides a kind of biological colorimetric sensor and preparation method thereof, based on Asia
Methyl blue interacts with single stranded DNA and utilizes the Conformation Transition for the single stranded DNA for carrying poly- cytimidine to build.
Present invention also offers a kind of application of biological colorimetric sensor detection glucose, the glucose sensing of preparation is utilized
Device, the ultraviolet and visible absorption peak value that the glucose solution of various concentrations is formed is different, is embodied in the original allusion quotation of methylene blue
Regular change occurs for type absworption peak and the ssDNA new absworption peak to be formed that interacted with methylene blue.Based on this
Operation principle, concentration of glucose can build linear relationship by colorimetric method and the signal detection of ratio, realize and glucose is passed
Sensor sensitivity, specific detection.
A kind of preparation method of biological colorimetric sensor provided by the invention, comprises the following steps：
The mixture of glucose solution and glucose oxidase solution is added in cushioning liquid, cultivates, then adds
Methylene blue solution, mix, add ssDNA cushioning liquid, mixed solution is made；Produce biological colorimetric sensor.
The preparation method of the ssDNA cushioning liquid is：The 2.5OD of purchase ssDNA is dissolved in Tris-EDTA bufferings
The DNA cushioning liquid that concentration is 100 μM is obtained in solution, is saved backup at 4 DEG C；
Tris-EDTA cushioning liquid Tris containing 10mM, 1mM EDTA, and pH 7-9, preferable pH 8.0.
Further, the ssDNA sequences are CCCCCCCCCCCCCCCCCCCCCCCC (poly24C).
Further, the condition of the culture is 25 DEG C of -35 DEG C of culture 30mim-60min, and preferably 25 DEG C are cultivated
30min。
Further, the mixture of glucose solution and glucose oxidase solution is added in cushioning liquid, cultivated,
The pH value of solution is reduced using the gluconic acid of reaction generation, induction ssDNA is folded into a close four overlapping structures
Referred to as " i-motif " DNA, the interaction of ssDNA and methylene blue can be hindered to varying degrees, so as to change ssDNA
The principle for the ultraviolet and visible absorption peak value to be formed that interacted with methylene blue, is prepared biological colorimetric sensor.
A kind of biological colorimetric sensor, is prepared using the above method.
A kind of application of biological colorimetric sensor detection glucose provided by the invention.
Detection method is：
Take the glucose solution of various concentrations gradient to be added separately in Tris-EDTA cushioning liquid, then be separately added into Portugal
Grape oxidase solution, culture；Methylene blue solution is then respectively adding, then is separately added into ssDNA cushioning liquid, is mixed
Solution, solution colour is observed, detect ultraviolet-visible absorption spectroscopy；The linear relationship of ratio absorbance and different concentration of glucose,
Realize the detection to glucose.
Further, in the mixed solution, methylene blue is 25 with ssDNA mol ratios:1.
Further, the condition of the culture is 25 DEG C of -35 DEG C of culture 30mim-60min, and preferably 25 DEG C are cultivated
30min。
Specifically detection method is：
The glucose solution of 20 μ L concentration gradients is taken to be added separately to 108 μ L Tris-EDTA (10mM Tris, 1mM
EDTA, pH 8.0) in cushioning liquid, then 20 μ L 3mM glucose oxidase solutions are separately added into, 25 DEG C of -35 DEG C of culture 30mim-
60min；50 μ L, 100 μM of methylene blue solutions are then respectively adding, 2 μ L, 100 μM of ssDNA cushioning liquid is added, is mixed
Solution is closed, observes solution colour, detects ultraviolet-visible absorption spectroscopy；The linear pass of ratio absorbance and different concentration of glucose
System, realizes the detection to glucose.
The ultraviolet and visible absorption peak value different manifestations that the present invention is formed using the glucose solution of various concentrations are methylene
Regular change occurs for blue original typical absorption peak and the ssDNA new absworption peak to be formed that interacted with methylene blue
Change.Based on this operation principle, concentration of glucose can build linear relationship by colorimetric method and the signal detection of ratio, realize
To glucose sensor sensitivity, specific detection.
The glucose sensor of Conformation Transition structure provided by the invention based on the single stranded DNA for carrying poly- cytimidine, can
Detection applied to glucose.Present invention selection methylene blue goes to explore the interaction with single stranded DNA as molecular probe.
Single stranded DNA maintains original single-stranded structure efficiently to be interacted with methylene blue in neutral or alkaline environment.In addition
In the solution of one addition glucose and glucose oxidase mixture, reacting the gluconic acid of generation reduces the pH value of solution
Induction single stranded DNA is folded into a close four overlapping structures and is referred to as " i-motif " DNA, can hinder to varying degrees single
The interaction of chain DNA and methylene blue, inhaled so as to change single stranded DNA and the methylene blue UV, visible light to be formed that interacts
The principle of peak value is received, is realized to glucose sensitivity, specific detection.
Compared with prior art, the preparation method of glucose sensor provided by the invention, cost is low, and design is simple, no
Glucose with concentration can make solution colour change therefore easily with the naked eye read.Pass through methylene blue and single stranded DNA phase
Interaction and the Conformation Transition for utilizing the single stranded DNA for carrying poly- cytimidine, the sensor of detection glucose can be prepared.As a result
Show that this sensor has sensitive detection for 5 μM to concentration of glucose to 0.8mM, and there is simple to operate, high sensitivity, test limit
It is low.
Brief description of the drawings
Fig. 1 is that ssDNA conversions of occurred conformation under the induction of the mixture of glucose and glucose oxidase hinder and Asia
The interaction of methyl blue is to detect the schematic diagram of glucose；
Fig. 2A is the UV absorption visible absorption spectra of methylene blue (a) and ssDNA/ methylene blues (b)；
Fig. 2 B be ssDNA/ methylene blues (a) add glucose oxidase (b), hydrogen peroxide (c), glucose (d) with
And the common ultraviolet-visible absorption spectroscopy for adding glucose and glucose oxidase (e)；
Fig. 3 A are that different pH value correspond to ultraviolet-visible absorption spectroscopy；
Fig. 3 B are curve map of the ratio absorbance from different pH value；
Fig. 4 A are that the glucose of various concentrations corresponds to ultraviolet-visible absorption spectroscopy；
Fig. 4 B are linear relationship of the ratio absorbance from different concentration of glucose；
Fig. 5 is that (fructose, lactose, sucrose, dopamine, vitamin C, the ratio of uric acid and glucose are inhaled for the detection of selectivity
Luminosity).
Embodiment
Verify methylene blue and ssDNA interaction：
SsDNA sequences are dissolved in Tris-EDTA (10mM Tris, 1mM EDTA, pH 8.0) cushioning liquid, obtain 100
μM ssDNA cushioning liquid.Wherein, ssDNA sequences are CCCCCCCCCCCCCCCCCCCCCCCC (poly24C).
50 μ L, 100 μM of methylene blue solutions are taken to be added to 148 μ LTris-EDTA (10mM Tris, 1mM EDTA, pH
8.0) in cushioning liquid, 2 μ L ssDNA cushioning liquid are then added, obtain mixed solution, observe solution colour, detection is ultraviolet can
See absorption spectrum.
SsDNA keeps original single-stranded knot in Tris-EDTA (10mM Tris, 1mM EDTA, pH 8.0) cushioning liquid
Structure can efficiently act on methylene blue.The color of solution is purple, in ultraviolet-visible absorption spectroscopy figure, the allusion quotation of methylene blue
Type absworption peak declines, and new absworption peak occurs.
Embodiment 1
A kind of preparation method of biological colorimetric sensor, comprises the following steps：
50 μ L 20mM glucose solutions are taken to be added to 78 μ L Tris-EDTA (10mM Tris, 1mM EDTA, pH 8.0)
Cushioning liquid, 20 μ L 3mM glucose oxidase solutions are added, 25 DEG C of culture 30min, then add 50 μ L, 100 μM of methylenes
Base indigo plant solution, mix, add 2 μ L, 100 μM of ssDNA cushioning liquid, obtain mixed solution, observe solution colour, biological ratio
Colour sensor, detect ultraviolet-visible absorption spectroscopy.
Due to addition glucose and the mixture of glucose oxidase, reacting the gluconic acid of generation reduces the pH of solution
Value induction ssDNA is folded into a close four overlapping structures and is referred to as " i-motif " DNA, can hinder to varying degrees
SsDNA and methylene blue interaction, solution colour are changed into blueness from purple.In ultraviolet-visible absorption spectroscopy figure, methylene
Blue typical absorption peak reappears, and the new peak of formation declines.
Embodiment 2
The pH differences of cushioning liquid are controlled, other steps obtain different pH value and correspond to ultravioletvisible absorption light with embodiment 1
Spectrum；See Fig. 3 A, Fig. 3 B.
Embodiment 3
A kind of application of biological colorimetric sensor detection glucose, detects the application of glucose.
Specifically detection method is：
1) 0.05,0.1,1,2.5,3,4,5,8,10 and 20mM Portugal, by glucose dissolving in deionized water, are configured to
Grape sugar juice.
2) glucose solution of the concentration gradient of the 20 above-mentioned preparations of μ L, is taken to be added separately to 108 μ LTris-EDTA (10mM
Tris, 1mM EDTA, pH 8.0) in cushioning liquid, then 20 μ L 3mM glucose oxidase solutions are separately added into, 25 DEG C of cultures
30min；
3) 50 μ L, 100 μM of methylene blue solutions, are separately added into again, after mixing, then are separately added into 2 μ L100 μM ssDNA and are delayed
Solution is rushed, obtains mixed solution, observes solution colour, detects ultraviolet-visible absorption spectroscopy.Concrete outcome such as Fig. 4 A.
The pH value induction ssDNA that glucose and the gluconic acid of glucose oxidase reaction generation reduce solution is folded into
" i-motif " structure, understands the interaction for hindering ssDNA and methylene blue to varying degrees, and the glucose of various concentrations is molten
The ultraviolet and visible absorption peak that liquid is formed is different, is embodied in：The original typical absorption peak of methylene blue and ssDNA and methylene
Regular change occurs for the new absworption peak that the interaction of base blue phase is formed.Based on this operation principle, concentration of glucose can be with
By colorimetric method and the signal detection of ratio, linear relationship is built, such as Fig. 4 B；Realize to glucose sensor sensitivity, specifically
The detection of property.
Embodiment 4
Same method detects to fructose, lactose, sucrose, dopamine, vitamin C, uric acid and glucose respectively, knot
Fruit such as Fig. 5, illustrate that method choice provided by the invention is good.
Claims (8)
1. a kind of preparation method of biological colorimetric sensor, it is characterised in that the preparation method comprises the following steps：
The mixture of glucose solution and glucose oxidase solution is added in cushioning liquid, cultivates, then adds methylene
Base indigo plant solution, mix, add ssDNA cushioning liquid, mixed solution is made；Produce biological colorimetric sensor.
2. preparation method according to claim 1, it is characterised in that the ssDNA sequences are
CCCCCCCCCCCCCCCCCCCCCCCC(poly24C)。
3. preparation method according to claim 1 or 2, it is characterised in that the condition of the culture is 25 DEG C -35 DEG C and cultivated
30mim-60min。
4. preparation method according to claim 1 or 2, it is characterised in that the preparation method of the ssDNA cushioning liquid
For：The 2.5OD of purchase ssDNA is dissolved in the DNA cushioning liquid for obtaining that concentration is 100 μM in Tris-EDTA cushioning liquid,
Saved backup at 4 DEG C.
5. a kind of biological colorimetric sensor, it is characterised in that be prepared using the method described in claim any one of 1-4.
A kind of 6. application of the biological colorimetric sensor detection glucose described in claim 5.
7. application according to claim 6, it is characterised in that detection method is：
Take the glucose solution of various concentrations gradient to be added separately in Tris-EDTA cushioning liquid, then be separately added into glucose
Aoxidize enzyme solutions, culture；Methylene blue solution is then respectively adding, then is separately added into ssDNA cushioning liquid, obtains mixing molten
Liquid, solution colour is observed, detect ultraviolet-visible absorption spectroscopy；Ratio absorbance and the linear relationship of different concentration of glucose, reality
Now to the detection of glucose.
8. application according to claim 7, it is characterised in that in the mixed solution, methylene blue and ssDNA mol ratios
For 25:1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112326757A (en) * | 2020-06-22 | 2021-02-05 | 宁波大学 | Novel electrochemical biosensing method for detecting glucose oxidase and urease and application thereof |
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| CN101655493A (en) * | 2008-08-20 | 2010-02-24 | 中国科学院成都有机化学有限公司 | Colorimetric analysis method for measuring content of glucose and activity of glucose oxidase |
| CN103760161A (en) * | 2014-01-25 | 2014-04-30 | 福州大学 | Colorimetric detection method for glucose |
| CN104330393A (en) * | 2014-11-04 | 2015-02-04 | 福建医科大学 | Method for determining glucose by using gold nano-cluster as fluorescence probe |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112326757A (en) * | 2020-06-22 | 2021-02-05 | 宁波大学 | Novel electrochemical biosensing method for detecting glucose oxidase and urease and application thereof |
| CN112326757B (en) * | 2020-06-22 | 2022-12-02 | 宁波大学 | Electrochemical biosensing method for detecting glucose oxidase and urease |
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Application publication date: 20180109 |